Viral antigens

ABSTRACT

The present invention relates to a vaccine composition comprising VLPs containing L1 proteins or functional L1 protein derivatives from HPV 16, HPV 18, HPV 31 and HPV 45 genotypes.

This application is a continuation of application Ser. No. 11/746,841,filed May 10, 2007, pending, which is a continuation of application Ser.No. 10/508,222, which is a §371 of International Application No.PCT/EP03/02826, filed Mar. 17, 2003, now U.S. Pat. No. 7,217,419, thedisclosures of which are incorporated by reference herein. Thisapplication also claims benefit of the filing date of the Great BritainApplication No. 0206360.0, filed Mar. 18, 2002.

The present invention relates to vaccines against HPV. In particular theinvention relates to vaccines comprising virus like particles (VLPs),especially virus like particles comprising proteins from human papillomavirus (HPV).

Papillomaviruses are small DNA tumour viruses, which are highly speciesspecific. So far, over 100 individual human papillomavirus (HPV)genotypes have been described. HPVs are generally specific either forthe skin (e.g. HPV-1 and -2) or mucosal surfaces (e.g. HPV-6 and -11)and usually cause benign tumours (warts) that persist for several monthsor years. Such benign tumours may be distressing for the individualsconcerned but tend not to be life threatening, with a few exceptions.

Some HPVs are also associated with cancers. The strongest positiveassociation between an HPV and human cancer is that which exists betweenHPV-16 and HPV-18 and cervical carcinoma. Cervical cancer is the mostcommon malignancy in developing countries, with about 500,000 new casesoccurring in the world each year. It is now technically feasible toactively combat primary HPV-16 infections, and even establishedHPV-16-containing cancers, using vaccines. For a review on the prospectsfor prophylactic and therapeutic vaccination against HPV-16 see CasonJ., Clin. Immunother. 1994; 1(4) 293-306 and Hagenesee M. E., Infectionsin Medicine 1997 14(7) 555-556, 559-564.

Although minor variations do occur, all HPVs genomes described have atleast eight early genes, E1 to E8 and two late genes L1 and L2. Inaddition, an upstream regulatory region harbors the regulatory sequenceswhich appear to control most transcriptional events of the HPV genome.

HPV L1 based vaccines are disclosed in WO94/00152, WO94/20137,WO93/02184 and WO94/05792. Such a vaccine can comprise the L1 antigen asa monomer, a capsomer or a virus like particle. Methods for thepreparation of VLPs are well known in the art, and include VLPdisassembly-reassembly approaches to provide enhanced homogeneity, forexample as described in WO9913056 and U.S. Pat. No. 6,245,568. Suchparticles may additionally comprise L2 proteins. L2 based vaccines aredescribed, for example, in WO93/00436. Other HPV vaccines are based onthe Early proteins, such as E7 or fusion proteins such as L2-E7.

Despite the work on HPV vaccines there is still no broadly effectivevaccine against cervical cancer.

The present invention relates to an improved vaccine against humanpapilloma virus.

In a first aspect the present invention relates to a vaccine compositioncomprising VLPs containing L1 proteins or functional L1 proteinderivatives from HPV 16, HPV 18, HPV 31 and HPV 45.

The invention also relates to a method of vaccine production, the methodcomprising combining VLPs containing L1 proteins or functional L1protein derivatives from HPV 16, HPV 18, HPV 31 and HPV 45.

The invention further relates to use of a mixture of VLPs containing L1proteins or functional L1 protein derivatives from HPV 16, HPV 18, HPV31 and HPV 45 in the preparation of a vaccine for the prevention ofcervical cancer.

The invention further relates to a method of preventing cervical cancer,the method comprising delivering to an individual at risk of cervicalcancer an effective amount of a vaccine as described above, such as avaccine comprising a mixture of HPV 16, HPV 18, HPV31 and HPV 45 VLPs.

The VLPs of the present invention can be formed from either the fulllength HPV L1 protein or certain L1 derivatives using standard methodsin the art, for example as disclosed in WO99/13056 incorporated hereinby reference.

It is preferred that the L1 protein used to form the VLP is a truncatedL1 protein. Preferably at least one of the VLPs comprises a truncated L1protein, and preferably all the L1 proteins in the combination vaccineare truncated L1 proteins. Preferably the truncation removes a nuclearlocalisation signal. Preferably the truncation is a C-terminaltruncation. Preferably the C-terminal truncation removes fewer than 50amino acids, more preferably fewer than 40 amino acids. Most preferablythe C terminal truncation removes 34 amino acids from HPV 16 and 35amino acids from HPV 18.

Truncated L1 proteins are suitably functional L1 protein derivatives.Functional L1 protein derivatives are capable of raising an immuneresponse (if necessary, when suitably adjuvanted), said immune responsebeing capable of recognising a VLP consisting of the full length L1protein and/or the HPV type from which the L1 protein was derived.

VLPs of the invention may also comprise other types of functionalprotein derivatives, including mutants of the full length or truncatedHPV L1 proteins such as deletion, substitution, or insertion mutants.Suitable derivatives also include codon optimised sequences. The L1protein or derivative may also be a fusion protein, such as the fusionof the L1 protein with L2 or an early protein. Preferably fusionproteins comprise proteins from only one HPV genotype. VLPs made fromchimaeric L1 proteins in which L1 proteins from one genotype are linkedto L1 proteins from other genotypes are not preferred.

The L1 protein or functional protein derivative is suitably able to forma VLP, and VLP formation can be assessed by standard techniques such as,for example, electron microscopy and dynamic laser light scattering.

Preferably the polydispersity of the VLPs is less than 0.15, mostpreferably less than 0.1 and more preferably less than 0.08 whenmeasured using a Malvern Zetasizer 3000HS under conditions as describedherein.

Use of the term ‘protein’ or reference to a specific protein e.g. ‘L1’is hereinafter taken to include reference to functional proteinderivatives, unless otherwise indicated or obviously apparent from thecontext.

In a preferred aspect of the invention the vaccine of the invention hasonly four VLP types —HPV 16, HPV 18, HPV 31 and HPV 45 VLPs. Preferablythe VLPs are L1-only VLPs from each of these 4 genotypes.

Alternatively, and most preferred, the vaccine comprises an additionalHPV valency, making a pentavalent vaccine. Preferably the additionalvalency is a VLP comprising an L1 protein or functional derivative, asabove, from one of HPV 52, 53, 58, 33, 35, 56, and 59. Preferably the5^(th) genotype is HPV 33 when the vaccine is for use in South Americaor HPV 52, 53 or 58 when the vaccine is for use in Asia.

The present invention also extends to vaccines comprising 2 or moreadditional valencies, to provide a vaccine with 6 or more genotypes.

In one preferred embodiment the combination excludes VLPs from HPV 6a,6b or HPV 11 genotypes.

Preferably the vaccine of the invention is at least 55% effective inpreventing cervical cancer, more preferably 60%, 65%, 70%, 75%preferably 80% or even more effective in prevention of cervical cancer.For the avoidance of doubt, % efficacy in prevention of cervical cancermeans protection against all cervical cancer induced by HPV infection,and not just protection against cancer caused by one genotype.Prevention may be suitably assessed over 1 year post initialvaccination, although preferred vaccines are equally effective over 2,3, 4, 5 or more years. The % efficacy can be increased by selectingappropriate HPV genotypes to target the vaccine formulation to specificgeographical areas.

Preferably the combination of VLPs within the vaccine does not reducethe immunogenicity of each VLP type. In particular it is preferred thatthere is no interference between HPV VLPs in the combination of theinvention, such that the combined VLP vaccine of the invention is ableto offer effective protection against infection by each HPV genotyperepresented in the vaccine. Suitably the immune response against a givenVLP type in the combination is at least 50% of the immune response ofthat same VLP type when measured individually, preferably 100% orsubstantially 100%. For responses to the HPV 16 and HPV 18 VLPs, thecombined vaccine of the invention preferably stimulates an immuneresponse which is at least 50% of that provided by a combined HPV 16/HPV18 VLP vaccine. Suitably the immune response generated by the vaccine ofthe invention is at a level in which the protective effect of each VLPtype is still seen. The immune response may suitably be measured, forexample, by antibody responses, as illustrated herein.

The vaccine of the invention may be used to treat or prevent HPVinfection and/or disease. For example the vaccine may be usedtherapeutically to reduce viral load and/or infections that lead tocervical carcinoma or CIN III sequelae. The invention thus relates touse of the vaccine of the invention in the therapeutic treatment ofdiseases related to HPV infection and in prophylaxis of infection ordisease. The invention also relates to use of the VLP combination of theinvention in generation of an immune response against HPV 16, 18, 31 and45.

The vaccine of the invention may optionally be formulated with VLPswhich provide protection against genital warts, such as VLPs containingL1 protein from HPV 6a, 6b and/or HPV 11 genotypes.

Preferably the VLPs comprise the HPV L1 protein only and no L2 proteinor protein fragment.

Vaccines of the invention may comprise other proteins or proteinfragments in addition to the L1 protein or derivative. Proteins/peptidesmay be delivered in chimaeric form with the L1 protein in the VLP,encapsulated within a VLP or co-formulated in a mixture with the VLP's.Other proteins or peptides may also be co-administered with the vaccineof the invention.

In one aspect the vaccine comprises an HPV L2 protein or L2 derivativesuch as an L2 peptide, for example as disclosed in K. Kawana et alVaccine 19, (2001) p 1496-1502, incorporated herein by reference. In afurther preferred embodiment the vaccine of the invention may beformulated with HPV early antigens such as E1, E2, E3, E4, E5, E6, E7,E8 or immunologically active derivatives thereof. When delivered inchimaeric form it is preferable to utilise an immunogenic fragment ofabout 30-60 amino acids of the early antigen.

Optionally the vaccine may also be formulated or co-administered withnon-HPV antigens. Suitably these antigens can provide protection againstother diseases, most preferably sexually transmitted diseases such asherpes simplex virus, chlamydia and HIV. We particularly prefer that thevaccine comprises gD or a truncate thereof from HSV, preferably the gD2tprotein as described in WO 99/45957. In this way the vaccine providesprotection against both HPV and HSV. Preferred HIV antigens aredescribed in WO/9916884 and WO/0154719.

The present invention generally relates to a mixture of VLPs containingcapsid proteins from HPV16, 18, 31 and 45, such as L1-only VLPs.

In a particularly preferred embodiment, the invention provides a vaccinecomprising a mixture of HPV 16 VLPs, HPV 18 VLPs, HPV 31 VLPs and HPV 45VLPs. Reference herein to ‘HPV 16 VLP’, for example, is a reference toan L1 VLP wherein the L1 protein or L1 derivative is from HPV 16. Thesame nomenclature principle applies, by extension, to other VLPsdescribed herein, such as HPV 18, HPV 31 and HPV 45 VLPs.

Preferably each VLP contains L1 protein from only 1 HPV genotype. Such avaccine may be formulated by production of individual VLPs from HPV 16,18, 31 and 45, followed by combination of such VLPs. Preferably thereare no other HPV proteins in the VLP other than L1.

Also preferred are VLPs containing proteins from only one HPV genotype,such as VLPs with L1 and L2 from HPV 16.

However, in an alternative embodiment of the invention, the VLPs may bemixed VLPs, a mixed VLP comprising L1 protein from one genotype incombination with L1 protein from a second genotype, wherein thedifferent L1 proteins are not chimaeric L1 proteins, but associatetogether within the same capsid structure to form immunogenic VLPs.

Preferred combinations include any permutation of genotypes 16, 18, 31and 45—for example, the invention may comprise a mixed HPV 16/HPV 18 VLPin combination with a mixed HPV31/HPV 45 VLP, or mixed 16/31 VLPS incombination with mixed 18/45 VLPs. Combinations of more than 2 L1genotypes within 1 VLP are also contemplated.

Mixed VLPs may be produced by separate expression of the individual L1proteins followed by combination to form VLPs, as exemplified herein.Alternatively multiple L1 proteins may be expressed within the samecell, from one or more DNA constructs. For example, multiple DNAconstructs may be transformed or transfected into host cells, eachvector encoding a different L1 protein. Alternatively a single vectorhaving multiple L1 genes, controlled by a shared promoter or multipleindividual promoters, may be used. IRES elements may also beincorporated into the vector, where appropriate. Using such expressionstrategies the co-expressed L1 proteins may be co-purified forsubsequent VLP formation, or may spontaneously form mixed VLPs which canthen be purified.

Where mixed VLPs are used, a preferred process for mixed VLP productioncomprises preparation of VLP L1 proteins or derivatives, such as L1proteins, from different papillomavirus genotypes, mixing the proteinsif necessary and assembly of the proteins to produce mixed VLPs. The L1proteins may be in the form of a crude extract, be partially purified orpurified prior to mixing. Preferably the proteins are at least partiallypurified before being combined. Optionally, further purification of themixed VLPs may be carried out after assembly. Where additional antigensare used, then these may be added where appropriate.

In one embodiment mixed VLPs may be made by disassembly of 2 or moreVLPs, followed by combination of the disassembled VLP components at anysuitable point prior to reassembly. This approach is suitable when VLPsspontaneously form when the L1 protein is expressed, as occurs forexample, in some yeast strains. Where the expression of the L1 proteindoes not lead to spontaneous VLP formation, preparations of L1 proteinsor capsomers may be combined before assembly into VLPs.

Assembly of VLPs is generally achieved by removal of a reducing agent.As such, in mixed VLP production, the mixing of proteins preferablytakes place prior to the removal of a reducing agent from the mixture ofproteins. Preferably the production of mixed VLPs comprises the step ofmixed VLP formation from a mixture of dissociated L1 proteins by removalof a reducing agent from the mixture under conditions that allow VLPs toform.

Preferably the reassembly process results from removal of a reducingagent such as mercaptoethanol.

It is, however, known that VLP formation is dependent upon pH, metalions and salinity as well as the presence of a reducing agent. As such,under certain circumstances, it may be envisaged that VLPs might form inthe presence of a reducing agent. It is only important to the inventionthat mixing of the proteins from different genotypes occurs prior to thechange in environmental condition that allows the mixed VLPs to form,whether this is pH, metal ions, salinity, reducing environment orcombination of these.

Where mixed VLPs are used, preferably the components of the VLPs aremixed in the proportions in which they are desired in the final mixedVLP. For example, a mixture of the same amount of a partially purifiedL1 protein from HPV 16 and HPV 18 provides a mixed VLP withapproximately equal amounts of each protein.

Vaccine solutions comprising mixed VLPs may be stabilised bycompositions known in the art, such as those of WO 98/44944, WO0045841,incorporated herein by reference.

For all vaccines of the invention, it is preferred that the vaccine isused for the vaccination of adolescent girls aged 10-15, preferably10-13 years. The vaccine may also be administered to women following anabnormal pap smear or after surgery following removal of a lesion causedby HPV.

Preferably the vaccine is delivered in a 2 or 3 dose regime, for examplein a 0, 1 month regime or 0, 1 and 6 month regime respectively. Suitablythe vaccination regime incorporates a booster injection after 5 to 10years, preferably 10 years.

Preferably the vaccine is a liquid vaccine formulation, although thevaccine may be lyophilised and reconstituted prior to administration.

The vaccines of the invention may also comprise adjuvants in combinationwith the VLPs. Suitably the VLPs of the invention are used incombination with aluminium, and are suitably adsorbed or partiallyadsorbed onto aluminium adjuvants. Also preferred are adjuvants whichstimulate a Th1 type response such as 3DMPL or QS21. Suitably theadjuvant is an aluminium salt, preferably in combination with 3D MPL,such as aluminium phosphate and 3D-MPL.

A preferred adjuvant is aluminium hydroxide, with the combination ofaluminium hydroxide with 3D-MPL especially preferred.

When VLPs are adsorbed on to aluminium containing adjuvants, theadjuvant is preferably added before mixing of the VLPs to form the finalvaccine product.

The vaccine may also comprise aluminium or an aluminium compound as astabiliser, and the present invention also relates to a stabilisedcombination vaccine wherein the VLPs are adsorbed onto an aluminiumsalt. Suitably the VLPs are more stable over time after adsorption ontoan aluminium salt than in the absence of aluminium. Preferablystabilised VLPs are obtained or obtainable by methods according toexample 1 section C3.

The vaccines of the invention may be provided by any of a variety ofroutes such as oral, topical, subcutaneous, musosal (typicallyintravaginal), intraveneous, intramuscular, intranasal, sublingual,intradermal and via suppository. Intramuscular and intradermal deliveryare preferred.

The dosage of VLP and other proteins will vary with the condition, sex,age and weight of the individual, the administration route and HPV ofthe vaccine. The quantity may also be varied with the number of VLPtypes. Suitably the delivery is of an amount of VLP suitable to generatean immunologically protective response. Suitably each vaccine dosecomprises 1-100 μg of each VLP, preferably 5-80 μg, more preferably 5-30μg each VLP, most preferably 5-20 μg of each VLP with 5 μg, 6 μg, 10 g,15 μg or 20 μg especially preferred.

The multivalent vaccine of the present invention is suitably produced bycombining purified L1 VLPs. Methods for the production of L1 VLPs arewell known in the art, and include for example methods given inWO9531532, WO9615247, WO00/09671 and U.S. Pat. No. 5,888,526, the wholecontents of which are incorporated herein.

Suitably the VLPs of the invention are made by disassembly andreassembly of VLPs, to provide homogenous and pure VLPs. Examples ofsuitable processes are given in WO0057906, U.S. Pat. No. 6,245,568 andWO9913056.

Preferably the VLPs are prepared from insect cells such as Sf9 or Hi-5cells, although any suitable cells such as E. coli or yeast cells, forexample, S. cerevisiae S. pombe or Pichia sp. may also be used.

Preferably the purification of VLPs after L1 expression includes one ormore of the steps of anion exchange chromatography (Di methyl aminoethyl—DMAE), anion exchange chromatography (trimethyl amino ethyl—TMAE),hydroxyapatite chromatography, filtration such as nanometric filtrationor ultrafiltration, or hydrophobic interaction chromatography.Preferably at least one anion exchange step is performed duringpurification, and more preferably 2 anion exchange steps are used.Preferably at least one anion exchange purification step is performedprior to mixing the proteins. Optionally a UV irradiation step may beemployed.

For the avoidance of doubt, the entire teaching of all documentsreferred to herein is incorporated by reference.

The present invention is illustrated by the following non-limitingExamples and Figures, wherein:

FIG. 1 illustrates mixed VLPs in comparison with HPV 16 VLPs as assessedby EM;

FIGS. 2 and 3 illustrate size distribution of mixed VLPs;

FIG. 4 illustrates antibody responses against VLP 16 in a mixed HPV 16,18, 31, 45 combination vaccine vs. an HPV 16 control;

FIG. 5 illustrates antibody responses against VLP 18 in a mixed HPV 16,18, 31, 45 combination vaccine vs. an HPV 18 control;

FIG. 6 illustrates antibody responses against VLP 31 in a mixed HPV 16,18, 31, 45 combination vaccine vs. an HPV 31 control; and

FIG. 7 illustrates antibody responses against VLP 45 in a mixed HPV 16,18, 31, 45 combination vaccine vs. an HPV 45 control.

EXAMPLE 1

The combination of HPV 16 and HPV 18 L1 VLPs is detailed herein. L1proteins from other HPV genotypes may be readily produced by similarmethods, already known in the art.

A Preparation of HPV 16/18 L1 VLPs

Production of HPV 16 and HPV 18 VLPs was carried out using standardprotocols—for example, see WO9913056. HPV 16/18 proteins were expressedin Trichoplusia ni (High Five™) cells (at a density of ˜350000 cells/ml)infected with recombinant Baculovirus (MOI of 0.3) encoding the HPV 16or 18 L1 gene of interest. Cells were harvested approximately 72 hourspost infection.

B Cell Harvest/Antigen Extraction

The antigen (L1-16/18) was extracted from Hi5 cells in a three stepprocess of concentration, extraction, clarification. The concentrationstep consists removes up to 90% of the culture medium, and was performedby tangential flow filtration. The extraction step was performed with ahypotonic buffer (Tris 20 mM, pH 8.5). A volume equal to the culturevolume was used to perform the extraction. A contact time of minimumhalf an hour under smooth agitation was used. The clarification wasperformed by tangential flow filtration.

C Purification

The purification process was carried out at room temperature.β-mercaptoethanol (4% w/w) was added to the extract in order todisassemble the VLP's into capsomers, for both antigens, L1-16/18.Glycerol was added up to a concentration of w/w 10% just prior to theaddition of β-mercaptoethanol.

All buffers used were filtered on 0.22 μm filters prior to storage at 2°C.-8° C. Prior to each purification run, gel matrixes are sanitised andequilibrated with appropriate buffer before sample loading.

Purification regiemes are given for the separate purification of L1 fromboth HPV 16 and 18. These schemes are broadly similar, and involve thesteps of:

Anion exchange chromatography (Di methyl amino ethyl—DMAE),Anion exchange chromatography (trimethyl amino ethyl—TMAE),Hydroxyapatite chromatography,Nanometric filtration (Planova),

Ultrafiltration,

Hydrophobic interaction chromatography (using Octyl Sepharose) for HPV18 or Anion exchange chromatography (DEAE) for HPV 16; andSterile filtration.

Specifically: C1 Purification of L1-18 Antigen Anion ExchangeChromatography DMAE

The clarified extract (protein at a concentration of ˜1 g/ml, with theL1 protein at ˜150 mg/ml) is applied to an anion exchange column (DiMethyl Amino Ethyl). Elution is performed with (Tris 20 mM|NaCl 200mM|4% β-mercaptoethanol BME) buffer, pH 7.9±0.2. The antigen is elutedin approximately 5 column volumes and the elution profile is monitoredat 280 nm.

Anion Exchange Chromatography TMAE

The eluate of the first step is diluted with 1 volume of H₂O/BME 4%. Thediluted eluate is then applied to a second anion exchange column (TriMethyl Amino Ethyl). Elution is performed with (20 mM Tris|NaCl 200mM|4% BME) buffer, pH 7.9±0.2. The antigen is eluted in approximately 4column volumes and the elution profile is monitored at 280 nm.

Hydroxyapatite Chromatography

The eluate of the TMAE step is applied to a hydroxyapatite (HA) column.After sample application, the gel is eluted with approximately 2.5column volumes of (NaH₂PO₄ 100 mM|NaCl 30 mM|4% BME) buffer, pH 6.0±0.2.

Nanometric Filtration (Planova)

The HA eluate is diluted in order to reach the following conditions:(NaH₂PO₄ 25 mM|NaCl 10 mM|4% BME) buffer, pH 7.5±0.2.

Then it is filtered successively on a 0.2 μm prefilter and on a Planova15N filter of 0.12 m². The filtration is performed at constant pressure200 mbar±20 mbar.

Ultrafiltration

The ultrafiltration is performed with a tangential flow ultrafiltrationsystem equipped with polyethersulfone membranes (Centramate cassette 0.1m², 100 kD).

The Planova eluate is treated to reach the following conditions:(NaH₂PO₄ 100 M|NaCl 30 mM|4% BME), pH 6.0±0.2; then it is loaded in thesystem, concentrated 5 fold and dia-filtrated with continuous injectionof ˜10 starting volumes of (NaH₂PO₄ 20 mM|NaCl 500 mM) buffer, pH6.0±0.2.

Hydrophobic Interaction Chromatography (Octyl Sepharose)

The ultrafiltration permeate is applied to an Octyl Sepharose column.

This chromatography step is run in the negative mode with approximately5 column volumes of (Na₃PO₄ 20 mM|NaCl 500 mM) buffer, pH 6.0±0.2.

Sterile Filtration

The purified L1-18 antigen solution is sterilised by filtration on a0.22 μm membrane.

C2 Purification of L1-16 Antigen Anion Exchange Chromatography DMAE

The clarified extract is applied to an anion exchange column (Di MethylAmino Ethyl).

Elution is performed with (Tris 20 mM|NaCl 180 mM|4% BME) buffer, pH7.9±0.2. The antigen is eluted in approximately 4 column volumes and theelution profile is monitored at 280 nm.

Anion Exchange Chromatography TMAE

The eluate of the first step is diluted with 1 volume of H₂O/BME 4%. Thediluted eluate is then applied to a second anion exchange column (TriMethyl Amino Ethyl). Elution is performed with (20 mM Tris|NaCl 180mM|4% BME) buffer, pH 7.9±0.2. The antigen is eluted in approximately 5column volumes and the elution profile is monitored at 280 nm.

Hydroxyapatite Chromatography (HA)

The eluate of the TMAE step is applied to a HA column.

After sample application, the gel is eluted with approximately 3 columnvolumes of (NaH₂PO₄ 100 mM|NaCl 30 mM|4% BME) buffer, pH 6.0±0.2.

Nanometric Filtration (Planova)

The HA eluate is diluted in order to reach the following conditions:(NaH₂PO₄ 25 mM|NaCl 10 mM|4% BME) buffer, pH 7.5±0.2.

Then it is filtered successively on a 0.2 μm prefilter and on a Planova15N filter of 0.12 m². The filtration is performed at constant pressure200 mbar±20 mbar.

Ultrafiltration

The ultrafiltration is performed with a tangential flow ultrafiltrationsystem equipped with polyethersulfone membranes (Centramate cassette 0.1m², 100 kD).

The Planova eluate is treated to reach the following conditions:(NaH₂PO₄ 100mM|NaCl 30 mM|4% BME), pH 6.0±0.2; then it is loaded in thesystem, concentrated 5 fold and dia-filtrated with continuous injectionof ˜10 starting volumes of (NaH₂PO₄ 20 mM|NaCl 500 mM) buffer, pH6.0±0.2.

Anion Exchange Chromatography DEAE

The ultrafiltration eluate is adjusted to the conductivity of theequilibrium buffer, (Na₃PO₄ 20 mM|NaCl 250 mM), pH 6.0±0.2 and appliedon an anion exchange column (Di Ethyl Amino Ethyl).

Elution is performed with (NaH₂PO₄ 20 mM|NaCl 500 mM) buffer, pH6.0±0.2. The antigen is eluted in approximately 3 column volumes and theelution profile is monitored at 280 nm.

Sterile Filtration

The purified L1-16 antigen solution is sterilised by filtration on a0.22 μm membrane.

C3

Each VLP type is adsorbed independently to produce a concentratedadsorbed monovalent.

Preparation of VLP 16 Concentrated Adsorbed Monovalent:

60 μg of purified VLPs from HPV16 are adsorbed on 150 μg Al³⁺ fromAl(OH)₃, at a pH of 6.0±0.2, for one hour at room temperature withgentle stirring. This concentrated adsorbed monovalent is stored at +4°C. Adsorption is checked by centrifuging the preparation and quantifyingVLPs in the supernatant.

Preparation of VLP18 Concentrated Adsorbed Monovalent:

60 μg of purified VLPs from HPV18 are adsorbed on 150 μg Al³⁻ fromAl(OH)₃, at a pH of 6.0±0.2, for one hour at room temperature withgentle stirring. This concentrated adsorbed monovalent is stored at +4°C. Adsorption is checked by centrifuging the preparation and quantifyingVLPs in the supernatant.

D Final Vaccine Preparation:

Concentrated adsorbed monovalents prepared by the above method werecombined to form a suspension containing 20 μg each VLP per dose. Finalvaccine is stored at +4° C.

Addition of VLPs from HPV 31 and 45 at a concentration of 20 μg each VLPcompletes the tetravalent vaccine.

The combined adsorbed bulks, or individual adsorbed bulks, may befurther mixed with adjuvants such as 3D-MPL.

EXAMPLE 2 A Preparation of HPV 16/18 L1 VLPs

Production of HPV 16 and HPV 18 VLPs was carried out using standardprotocols—as above

B Formation of Mixed VLPs

The process of the invention involves disassembly and then reassembly ofthe HPV 16 and 18 VLPs such that the reassembly of HPV L1 16 and 18 iscarried out together to permit the formation of a mixed VLP.

The HPV 16 and 18 VLPs may be combined at any suitable point in theabove process prior to the point at which the VLPs are reassembled.

By way of example 2 specific strategies have been tested:

1 mixing of both antigens after the HA step. Based on the L1concentration in HA pools, the two components are mixed to reach anequal concentration of HPV16 and 18 to start the UF step. In this caseafter the ultrafiltration step an Octyl speharose step is performed asfor HPV 18 purification followed by a DEAE step as performed in the HPV16 procedure.2 mixing of both extracts and copurify. Based on the L1 concentration inExtracts, the two valences are mixed to reach an equal concentration ofHPV16 and 18 to start the DMAE step. Again, after the ultrafiltrationstep an Octyl speharose step is performed as for HPV 18 followed by aDEAE step as performed in the HPV 16 procedure.

HPV16-HPV18 VLP-Mixed at DMAE Step

The same flow sheet is applied but the mixing is performed at the DMAEstep instead of the UF step. The concentration used for elution at theanion exchange DMAE TMAE steps is 200 mM.

Results HPV16-HPV18 VLP-Mixed at UF Step

2 lots of mixed VLPs (lot numbers 31b165c and 31b166c) were produced bycombining HPV 16 and HPV 18 L1 proteins.

Purity by SDS-Page

The purity of the mixed VLP's was as good as both “classical” HPV 16 orHPV 18 bulks. The purity of the bulks was higher than 95%.

EM Data—FIG. 1

The EM of 31B165C (UF Retentate after maturation) was compared to aclassical HPV16 lot (39B122c). VLP's were well formed, homogeneous insize, without aggregation; some ribbons of capsomeres are present inboth experiments.

Size Distribution

The size distribution of the VLP's were determined using a MalvernZetasizer 3000 HS.

The samples were measured undiluted into a plastic cuvette for Malvernanalysis (800 μl/cuvette).

The technical conditions were:

-   -   laser wavelength: 532 nm,    -   laser power: 50 mW,    -   scattered light detected at 90°,    -   temperature: 25° C.,    -   duration: automatic determination by the software,    -   number: 3 consecutive measurements,    -   z-average diameter: by cumulants analysis,    -   size distribution: by the Contin method.        Classical results for HPV18 L1-VLP's are: 70-80 nm with good        polydispersity (<0.1)        Classical results for HPV16 L1-VLP's are: 60-70 nm with good        polydispersity (<0.1)

For mixed VLP's, the following results were obtained:

31 B165c: 85 nm with good polydispersity (0.08). VLP's are almostcompletely formed at the beginning of maturation31 B166c: 76 nm with good polydispersity (0.08).

The size distribution of lot 31 B165c and 31 B 166c as measured bydynamic laser light scattering is shown in FIGS. 2 and 3.

HPV16-HPV18 VLP Mixed at DMAE Step

Lot no. 31B167B was made up from lots E18L1C005 (HPV18) and 39B167(HPV16).

Purity by SDS-Page

The purity of the mixed VLP's was as good as both “classical” bulks.

The purity of the bulks was higher than 95%.

Size Distribution

The size distribution of the VLP's were determined by using a MalvernZetasizer 3000 HS.

Classical results for HPV18 L1-VLP's are: 70-80 nm with goodpolydispersity (<0.1)Classical results for HPV16 L1-VLP's are: 60-70 nm with goodpolydispersity (<0.1)

For mixed VLP's, the following results were obtained:

HPV16-HPV18 31B167B: 74 nm with good polydispersity (0.07). VLP's werealmost completely formed at the beginning of maturation

EXAMPLE 3 Production of a Mixed HPV 16, 18, 31, 45 Combination VaccineIntroduction

An immunogenicity study was performed in Balb/C mice using a combinationof C-terminally truncated L1 VLPs 16, 18, 31 & 45 adjuvanted withalum+3D-MPL (herein ‘adjuvant A’—50 μg aluminium salt and 5 μg 3D MPL)

4 groups of 10 mice were immunised twice intramuscularly on day 0 and 21respectively with:

1. VLP 31 (2 μg)/adjuvant A2. VLP 45 (2 μg)/adjuvant A3. VLP 16 (2 μg) and VLP 18 (2 μg)/adjuvant A4. VLP 16 (2 μg), VLP 18 (2 μg), VLP 31 (2 μg), VLP 45 (2 μg)/adjuvant A

Antibody responses against VLPs 16, 18, 31 and 45 were monitored on serataken at day 35 (14 days post dose II).

Results are shown in FIGS. 4-7

Antibody Response Against VLP 16

-   -   Strong antibody responses are induced in post II sera by either        VLP16 formulated with VLP 18 on adjuvant A (group 3) or by the        full combo (group 4)    -   Similar level of antibodies directed against VLP 16 are measured        in both groups and no interference is observed.

Antibody Response Against VLP 18

-   -   Strong antibody responses are induced in post II sera by either        VLP 18 formulated with VLP 16 on adjuvant A (group 3) or by the        full combo (group 4).    -   Similar level of antibodies directed against VLP 18 are measured        in both groups (less than 1.5 fold difference) and no        interference was observed.

Antibody Response Against VLP 31

-   -   Strong antibody responses are induced in post II sera by either        VLP31 formulated alone on adjuvant A (group 1) or by the full        combo (group 4).    -   Similar level of antibodies directed against VLP 31 are measured        in both groups, therefore no interference is observed.

Antibody Response Against VLP 45

-   -   Strong antibody responses are induced in post II by either VLP        45 formulated alone on adjuvant A (group 2) or by the full combo        (group 4).    -   Similar level of antibodies directed against VLP 45 are measured        in both groups, thus no interference is observed.

Conclusions

No interference is observed when the four VLPs (VLPs16, 18, 31 & 45) aredelivered as a combination.

1. A mixture of VLPs comprising VLPs containing L1 proteins orfunctional L1 protein derivatives from HPV 16, HPV 18, HPV 31 and HPV 45genotypes.
 2. A mixture of VLPs according to claim 1, comprising amixture of HPV 16 VLPs, HPV 18 VLPs, HPV 31 VLPs and HPV 45 VLPs, eachVLP comprising L1 proteins or functional L1 protein derivatives fromonly 1 HPV genotype.
 3. A mixture of VLPs according to claim 2, whereinthe VLPs are L1-only VLPs comprising HPV L1 protein or functional L1protein derivatives and no other HPV protein.
 4. A mixture of VLPsaccording to claim 1, comprising VLPs consisting essentially only ofproteins from HPV 16, HPV 18, HPV 31 and HPV
 45. 5. A mixture of VLPsaccording to claim 1, wherein at least one L1 protein is a truncated L1protein.
 6. A mixture of VLPs according to claim 1, wherein thecomposition comprises a VLP containing an L1 protein or functionalderivative thereof from an additional HPV genotype.
 7. A mixture of VLPsaccording to claim 6, wherein the additional VLP comprises an L1 proteinor functional L1 protein derivative selected from one of HPV 33, 52, 53,58, 35, 56, and
 59. 8. A mixture of VLPs according to claim 1,additionally comprising an HPV early antigen or immunologically activefragment thereof selected from E1, E2, E3, E4, E5, E6, E7 or E8.
 9. Amixture of VLPs according to claim 1, formulated with an antigen derivedfrom an organism causing a sexually transmitted disease.
 10. A mixtureof VLPs according to claim 9, wherein the antigen is an HSV antigen orimmunologically active fragment thereof.
 11. A mixture of VLPs accordingto claim 9, wherein the antigen is an HIV antigen or immunologicallyactive fragment thereof.
 12. A mixture of VLPs according to claim 9,wherein the antigen is a chlamydia antigen or immunologically activefragment thereof.
 13. A mixture of VLPs according to claim 1, comprisingan HPV L2 protein or fragment thereof.
 14. A mixture of VLPs accordingto claim 1, additionally comprising an adjuvant.
 15. A mixture of VLPsaccording to claim 14, wherein the adjuvant is an aluminium salt.
 16. Amixture of VLPs according to claim 15, wherein the aluminium salt isaluminium hydroxide.
 17. A mixture of VLPs according to claim 15,additionally comprising 3D-MPL.
 18. A mixture of VLPs according to claim1, wherein the VLPs contain L1 proteins which are not chimaeric.
 19. Amethod of vaccine production, the method comprising combining VLPscontaining L1 proteins or functional L1 protein derivatives from HPV 16,HPV 18, HPV 31 and HPV 45 to form a vaccine as claimed in any ofclaim
 1. 20. A method of prevention of HPV infection or treatment ofdisease related to HPV infection, the method comprising delivering to anindividual at risk of infection or disease an effective amount of avaccine composition according to claim 1.